Blood is a rich and readily accessible source for the detection of diagnostic markers and therapeutic targets in many human diseases. Currently, however, only a handful of plasma proteins are routinely used in the clinic for diagnostic purposes. It is generally established that these low-abundance proteins contain most of the useful biomarkers, including those that are produced by specific diseases such as cardiovascular diseases, neurological disorders, autoimmune diseases, and cancer, but these low abundance proteins are difficult to detect because they are often masked by high-abundance proteins, particularly serum albumin. In serum and plasma, the quantities of high-abundance proteins and some low-abundance proteins span over 10 orders of magnitude. For example, low-abundance proteins such as growth factors and cytokines are present in one millionth to one trillionth of the abundant proteins.
Serum albumin, the most abundant protein in serum typically present at 45-50 mg/ml, constitutes about 55% of total serum protein. Albumin functions as a scaffold for binding proteins, lipids, small molecules in the intracellular space and has been found to form associations with peptide hormones such as insulin and glucagon; serum amyloid A, interferons, bradykinin, the amino-terminal peptide of HIV-1, gp41, the 14-kDa fragment of streptococcal protein G, and others. In order to carry out such diverse functions, it is likely that serum albumin population is heterogeneous and contains many different albumin complexes. Technologies currently available such as two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) developed by O'Farrel (O'Farrel, P., (1975) J. Biol. Chem. 250: 4007-4021) do not allow separation of protein complexes because they were carried out under denaturing conditions which dissociated the serum albumin complexes.
Due to the wide range of protein concentrations and high structural complexity of the constituent proteins, analysis of the proteome of plasma and serum represents a challenge. At present, low-abundance proteins can only be detected for further analysis by removal of abundant proteins to decrease both dynamic concentration range and complexity.
One approach for removing the most abundant plasma proteins, yielding an enriched pool of low-abundance proteins, is immunoaffinity chromatography (Gallent, S R., (2008) Methods Mol. Biol. 421:53-60). Although this method has increased the number of detectable proteins in plasma/serum analysis, it has several major drawbacks. First, up to 90% of potential protein biomarkers are known to be associated with the highly abundant carrier proteins in blood such as serum albumin. Depletion of the high-abundance proteins often removes many potentially important marker proteins. In addition, some low-abundance proteins may be retained in the column through nonspecific binding, resulting in their loss from the flow-through fractions. Extensive sample handling also increases the chance of sample loss and protein degradation, resulting in substantial sample-to-sample variation. The depletion process is also time-consuming and the immunoaffinity columns are rather expensive.
Realizing that most of potential protein biomarkers are likely to be associated with the highly abundant carrier proteins in blood such as serum albumin, a different approach for partial enrichment of the low-abundance proteins is to capture serum albumin onto a solid support followed by selectively eluting the low-abundance proteins with solvents. However, serum albumin is composed of mixtures of complexes and selective removal of bound proteins based on affinity without first resolving the albumin complexes will have difficulty in obtaining the low-abundance proteins. Most of proteins detected by this procedure were abundant proteins present naturally and not related to the specific disease.
Diseases such as cancer are caused by, for example, DNA damage (i.e., mutation) in genes that regulate cell growth and division. It is often characterized by the production of abnormal proteins. Because of the difficulties indicated above it is virtually impossible to isolate the low-abundance disease proteins. Compounding the problem for their detection is the fact that in many diseased cells, these disease-associated proteins are degraded by proteolytic enzymes, generating peptide fragments that are subsequently released into the bloodstream. Being low molecular weight in nature, these peptide fragments generally have a half-life of only several hours and most of them are cleared from circulation by the kidney (Lowenthal et al. (2005) Clinical Chemistry 51: 1933-45). However, some of these peptide fragments have high affinity for serum albumin which has a rather long half-life of about 19 days. By their association with serum albumin to form complexes the longevity of these disease-related peptide fragments can be increased by approximately 60 to 100-fold (Dennis et al. (2002) J. Biol. Chem. 277: 35035-43).
Breast cancer is the second most common cause of cancer death in women in the United States and is also a cause of disability, psychological trauma, and economic loss. Breast cancer morbidity increases significantly if it is not detected early in its progression. Early detection of breast cancer before symptoms appear is highly desirable. Even so, it is estimated that between 15 to 25% of women with early stage breast cancer are currently missed by mammography particularly if they have dense breasts. The challenge is to address the inherent limitations of mammography by developing a simple blood test procedure that can detect early stage breast cancer and potentially enhance treatment and enhance the potential survival of the patient.